Unstable osmolality of microdrops cultured in non-humidified incubators.

PURPOSE: To investigate the stability of osmolality in non-humidified and humidified incubators for assisted reproductive technologies (ART). METHODS: Drops of three single-step culture media (media A, B, and C) were incubated for 5 or 6 days covered with four different mineral oils (oils A, B, C, and D) in non-humidified incubator A, non-humidified incubator B, or humidified incubator C to investigate the effects of incubator environment (humidification), drop volume, culture media, and mineral oil on the stability of osmolality in microdrops. RESULTS: A significant and linear increase was shown in the osmolality of 50-µL and 200-µL microdrops covered with mineral oil during 5 days incubation in non-humidified benchtop incubators. The maximum increase was 20 mOsm/kg, and the extent of the increase was affected by microdrop volume and possibly by the type of mineral oil used to cover the drops. In contrast, the osmolality of 50-µL and 200-µL microdrops did not change during 5 days incubation in a humidified benchtop incubator. CONCLUSIONS: Mineral oil alone may not adequately prevent gradual changes in the osmolality of low-volume microdrops during extended in vitro culture of human embryos in non-humidified incubators. As a result, the osmolality may increase to high enough levels to stress some human embryos and adversely affect clinical outcomes. We therefore recommend that the stability of osmolality should be given more consideration to ensure optimal culture conditions for ART.

Assessment of developmental potential of human single pronucleated zygotes derived from conventional in vitro fertilization.

PURPOSE: The aim of this study was to non-invasively validate the developmental potential of human single pronucleated (1PN) zygotes derived from conventional in vitro fertilization (c-IVF) at the zygote stage. METHODS: Fifty 1PN zygotes derived from 45 patients undergoing c-IVF were used. Immunohistochemistry and fluorescence live cell imaging were used to confirm normal chromosome segregation during the first mitosis. The usefulness of measuring pronuclear diameter was assessed on the basis of the presence or absence of a proper first cleavage and validated by subsequent development. RESULTS: Although approximately 80% (15/19) of 1PN zygotes contained a diploid genome, immunohistochemistry revealed an unequal distribution of paternal and maternal genomes at the first mitosis. Fluorescence live imaging revealed that 73% (8/11) of 1PN zygotes formed a functional mitotic spindle at the first mitosis resulting from diploid genomes, with 25% (2/8) of these forming a tripolar spindle. 1PN zygotes in which the pronucleus disappeared and that subsequently underwent cleavage had a pronuclear diameter ≥ 32.2 µm. The selection of 1PN zygotes based on pronuclear diameter resulted in zygotes that all formed mitotic spindles with poles during cleavage. Furthermore, 63% (5/8) of these zygotes reached the blastocyst stage. CONCLUSIONS: This study demonstrates the usefulness of a non-invasive assessment of 1PN zygotes derived from c-IVF as an indicator of developmental potential. Furthermore, diploid 1PN zygotes do not always exhibit normal chromosome segregation at the first mitosis. A pronuclear diameter ≥ 32.2 µm just before PN breakdown might be a useful criterion to assess 1PN zygotes that are capable of further development.

A Muscarinic Antagonist Reduces Airway Inflammation and Bronchoconstriction Induced by Ambient Particulate Matter in a Mouse Model of Asthma.

Ambient particulate matter (PM) can increase airway inflammation and induce bronchoconstriction in asthma. This study aimed to investigate the effect of tiotropium bromide, a long-acting muscarinic antagonist, on airway inflammation and bronchoconstriction induced by ambient PM in a mouse model of asthma. We compared the effect of tiotropium bromide to that of fluticasone propionate and formoterol fumarate. BALB/c mice were sensitized to ovalbumin (OVA) via the airways and then administered tiotropium bromide, fluticasone propionate, or formoterol fumarate. Mice were also sensitized to ambient PM via intranasal instillation. Differential leukocyte counts and the concentrations of interferon (IFN)-γ, interleukin (IL)-5, IL-6, IL-13, and keratinocyte-derived chemokine (KC/CXCL1) were measured in bronchoalveolar lavage fluid (BALF). Diacron-reactive oxygen metabolites (dROMs) were measured in the serum. Airway resistance and airway inflammation were evaluated in lung tissue 24 h after the OVA challenge. Ambient PM markedly increased neutrophilic airway inflammation in mice with OVA-induced asthma. Tiotropium bromide improved bronchoconstriction, and reduced neutrophil numbers, decreased the concentrations of IL-5, IL-6, IL-13, and KC/CXCL1 in BALF. However, tiotropium bromide did not decrease the levels of dROMs increased by ambient PM. Though eosinophilic airway inflammation was reduced with fluticasone propionate, neutrophilic airway inflammation was unaffected. Bronchoconstriction was improved with formoterol fumarate, but not with fluticasone propionate. In conclusion, tiotropium bromide reduced bronchoconstriction, subsequently leading to reduced neutrophilic airway inflammation induced by ambient PM.

Estimation of the effects of heavy Asian dust on respiratory function by definition type.

BACKGROUND: The adverse effects of Asian dust (AD) on health have been demonstrated in earlier studies, but there is no standardized definition for heavy-AD. This study aimed to examine which definition of heavy-AD has the most adverse effect on respiratory function. METHODS: One-hundred-and-thirty-seven adults with asthma, and 384 school children self-measured their morning peak expiratory flow (PEF). The four definitions of heavy-AD are: (1) the definition provided by the Japan Meteorological Agency (JMA), (2) daily median AD particle level ≥ 0.07 km-1, obtained through light detection and ranging (LIDAR) (3) hourly AD particle level ≥ 0.1 km-1, and (4) hourly level ≥ 0.07 km-1. Linear mixed models were used to estimate the effects of heavy-AD, by definition type, on daily PEF values. RESULTS: In adults with asthma, as per the JMA's definition, significantly reduced PEF were observed on heavy-AD days (lag 0), lag 0-1, and lag 0-3. In school children, after a heavy-AD event, as defined by the JMA, PEF significantly decreased on lag 0-1, lag 0-2, and lag 0-3. However, as per the other definitions, there was no significant decrease in the PEF in the adults and children. CONCLUSION: The associations between heavy-AD and respiratory function differed between these definitions.

Association between transported Asian dust and outdoor fungal concentration during winter in a rural area of western Japan.

BACKGROUND: Recently, Asian dust (AD) has become a serious health problem and several studies have clearly proven that AD can aggravate asthma. However, it remains unclear as to which components of AD have a strong effect on the asthma exacerbation caused by AD exposure. Outdoor fungi can increase emergency department visits and hospitalization for asthma exacerbation and can aggravate asthma symptoms. Therefore, this study was aimed at investigating the relationship between AD and outdoor fungi and determining the potential of fungi to cause airborne particulate matter (PM)-related inflammatory responses. METHODS: Airborne PM was collected each day from January 26, 2015 to February 27, 2015. Daily levels of outdoor fungi-associated PM were calculated using a culture-based method. Production of cytokines such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α was assessed in THP1 cells stimulated by the collected airborne PM each day. RESULTS: Daily levels of AD particles were assessed using Light Detection and Ranging and did not correlate with outdoor fungi (r = -0.17, P = 0.94). There was also no association between outdoor fungi and the daily production of IL-6 (r = 0.16, P = 0.37), IL-8 (r = 0.19, P = 0.30), or TNF-α induced by collected PM (r = 0.07, P = 0.70). However, the daily levels of AD particles were significantly associated with IL-6 (r = 0.91, P < 0.0001), IL-8 (r = 0.64, P = 0.0004), and TNF-α (r = 0.72, P < 0.0001) production. CONCLUSION: AD did not increase the acute levels of outdoor fungi and outdoor fungi did not affect the cytokine production induced by airborne PM. These results suggest that outdoor fungi do not have any detectable effect on the asthma exacerbation caused by AD exposure.

Association of Short-Term Exposure to Ambient Fine Particulate Matter with Skin Symptoms in Schoolchildren: A Panel Study in a Rural Area of Western Japan.

Numerous studies have unmasked the deleterious effects of particulate matter less than 2.5 µm (PM2.5) on health. However, epidemiologic evidence focusing on the effects of PM2.5 on skin health remains limited. An important aspect of Asian dust (AD) in relationship to health is the amount of PM2.5 contained therein. Several studies have demonstrated that AD can aggravate skin symptoms. The current study aimed to investigate the effects of short-term exposure to PM2.5 and AD particles on skin symptoms in schoolchildren. A total of 339 children recorded daily skin symptom scores during February 2015. Light detection and ranging were used to calculate AD particle size. Generalized estimating equation logistic regression analyses were used to estimate the associations among skin symptoms and the daily levels of PM2.5 and AD particles. Increases in the levels of PM2.5 and AD particles were not related to an increased risk of skin symptom events, with increases of 10.1 µg/m³ in PM2.5 and 0.01 km-1 in AD particles changing odds ratios by 1.03 and 0.99, respectively. These results suggest that short-term exposure to PM2.5 and AD does not impact skin symptoms in schoolchildren.

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number.

PURPOSE: Normally fertilized zygotes generally show two pronuclei (2PN) and the extrusion of the second polar body. Conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI) often result in abnormal monopronuclear (1PN), tripronuclear (3PN), or other polypronuclear zygotes. In this study, we performed combined analyses of the methylation status of pronuclei (PN) and the number of centrosomes, to reveal the abnormal fertilization status in human zygotes. METHOD: We used differences in DNA methylation status (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) to discriminate between male and female PN in human zygotes. These results were also used to analyze the centrosome number to indicate how many sperm entered into the oocyte. RESULT: Immunofluorescent analysis shows that all of the normal 2PN zygotes had one 5mC/5hmC double-positive PN and one 5mC-positive PN, whereas a parthenogenetically activated oocyte had only 5mC staining of the PN. All of the zygotes derived from ICSI (1PN, 3PN) had two centrosomes as did all of the 2PN zygotes derived from c-IVF. Of the 1PN zygotes derived from c-IVF, more than 50 % had staining for both 5mC and 5hmC in a single PN, and one or two centrosomes, indicating fertilization by a single sperm. Meanwhile, most of 3PN zygotes derived from c-IVF had a 5mC-positive PN and two 5mC/5hmC double-positive PNs, and had four or five centrosomes, suggesting polyspermy. CONCLUSIONS: We have established a reliable method to identify the PN origin based on the epigenetic status of the genome and have complemented these results by counting the centrosomes of zygotes.

Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica, against Melissococcus plutonius, the causal agent of European foulbrood disease.

European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee, Apis cerana japonica F. (Hymenoptera: Apidae), were characterized and evaluated for their potential to inhibit the growth of Melissococcus plutonius corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae), the causative agent of European foulbrood. Analysis of 16S rRNA gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to Bacillus, Staphylococcus, and Pantoea. The isolates were screened against the pathogenic bacterium M. plutonius by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus Bacillus exhibited inhibitory activity against M. plutonius. In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with M plutonius, suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.

Analysis of compaction initiation in human embryos by using time-lapse cinematography.

PURPOSE: To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. METHODS: One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. RESULTS: Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. CONCLUSIONS: The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

PURPOSE: To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. METHODS: We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. RESULTS: Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. CONCLUSIONS: The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

Trichostatin A, a histone deacetylase inhibitor, down-regulates interleukin-12 transcription in SV-40-transformed lung epithelial cells.

Inhibition of histone deacetylation results in increased gene expression. Trichostatin (Ts)A, a specific histone deacetylase (HDAC) inhibitor, up-regulates transcription of some genes but represses expression of others. We quantified histone acetylation in SV-40-transformed lung epithelial cells using flow cytometry. Further, to evaluate the effect of TsA on transcription of genes associated with airway inflammation, we measured interleukin (IL)-8 production by enzyme-linked immunosorbent assay as well as IL-12 transcription by reverse transcription-polymerase chain reaction, in the transformed cells after stimulation with lipopolysaccharide (LPS) in the presence of TsA. Pretreatment of cells with TsA before LPS stimulation induced hyperacetylation of histones (especially in the S phase of the cell cycle), enhanced IL-8 production, and suppressed IL-12p35 and IL-12p40 mRNA accumulation. Thus we have demonstrated a useful way to detect hyperacetylation at the single-cell level, as well as the ability of an HDAC inhibitor to repress genes in epithelial cells.